The invention relates to a process for the lysis of a culture of lactic acid bacteria, or a product containing such culture, by means of a lysin e.g. in producing a fermented food product, e.g. in cheese-making. Such a process is known from WO 90/00599 (AGRICULTURAL & FOOD RESEARCH COUNCIL (AFRC), M. J. Gasson, published Jan. 25, 1990, ref. 1). According to that patent specification the lysin from a Lactococcus (preferably prolate-headed) bacteriophage was used to lyse bacterial starter cultures during cheese-making. Exemplified was the lysin of the bacteriophage .phi.vML3 of Lactococcus lactis ML3. In particular, the lysin can be added to a cheese product or a cheese precursor mixture, e.g. after whey removal, milling and salting. However, this solution has the disadvantage that thorough mixing of the contents of the lysed cells with the cheese product is not easily obtained. Another disadvantage is that the lysin was produced by Escherichia coli cells, which are not food-grade. It is explicitly stated if the cell wall of the host cell is not itself degraded by the lysin then the lysin secreting transformed host may be useful in suppressing populations of bacteria which are susceptible to lysis by the lysin. Nothing is mentioned regarding addition of a transformed host cell in improving cheese flavor, certainly not a transformed lactic acid bacterium.
As an alternative it is suggested in that patent specification "to encapsulate the lysin so that the timing of its addition is not important. The encapsulating agent dissolves after the cheese-making process is complete thus not affecting the starter bacteria before their role in acidification was complete."
This suggested alternative has the disadvantages, that (a) an encapsulating material has to be used, and (b) said material must not dissolve before the end of the cheese making process. Moreover, if the encapsulated lysin is added at the beginning of the cheese-making process, e.g. while adding the cheese starter culture to the milk, about 90% of it is removed with the whey. Thus one has to add about tenfold the required effective amount, which is economically not attractive. In a later publication C. A. Shearman, K. Jury & M. J. Gasson (Feb. 1992, ref. 2) described an autolytic Lactococcus lactis expressing a cloned lactococcal bacteriophaze .phi.vML3 lysin gene. In particular they stated that
"(e)xpression of the cloned lysin did not impair the ability of Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris strains to metabolize lactose, to clot milk and produce acid (data not shown)".
It was suggested that during the exponential phase the lysin would not, or would insufficiently be expressed. It would only be expressed in sufficient amounts to lyse an appreciable proportion of the cells during the stationary phase, which occurs at the end of the normal fermentation process. The article illustrates that maintenance of transformed lactococcal strains could be a problem. Maintenance at a temperature below 30.degree. C. slightly delayed the onset of lysis but at 30.degree. C. regrowth of lysin resistant bacteria occurred. As alternative buffering in a sucrose medium with a sucrose percentage higher than 20% was given. This does not seem to be suitable in a process of fermentation like cheese making where the fermentation step occurs at 30.degree. C. or higher and the presence of more than 20% sucrose is not acceptable.
Furthermore, at the end of that publication it was indicated that expression in the stationary phase is not completely controlled. In addition the use of osmotic buffer in a cheese maturing process is probably not very efficient timewise. This can be illustrated by the length of time required for a Gouda cheese immersed in a brine bath to achieve the desired degree of salt flavour. For this cheese type for example the osmotic effect of salt concentration is not going to be very quick. The cheddar cheese making process would probably be more suitable as the salt addition step is more efficient, however, this still has the disadvantage of requiring a mixing step.
Both disclosures described the use of a lysin originating from a lactococcal bacteriophage lysin, that means an enzyme produced in nature by an undesired substance like a bacteriophage, because bacteriophage contaminations are a major problem in large scale industrial dairy fermentation processes.
In a review article R. Young (1992, ref. 4) gives a survey of the state of the art on bacteriophage lysis, both mechanism and regulation. Especially in the section "Lysis in Phage Infections of Gram-Positive Hosts" on pages 468-472 it was indicated that the DNA sequence found by Shearman c.s. (1989, ref. 5), which DNA sequence seems to be the same as that given in ref. 1, is probably not correct and that the deduced amino acid sequence might be quite different due to a mutation causing a phase shift in the reading frame.
In a publication of Ward c.s. (1993; ref. 6) it is also suggested that the sequence of Shearman et al. (1989; ref. 5) is probably not correct. Comparison with a very similar phage lysin gene confirmed that a frame shift in the Shearman et al. (ref. 5) sequence is needed for aligning the two DNA sequences. Moreover, this comparison teaches that the real phage lysin is encoded by an ORF that is probably 45 bases longer than disclosed by Shearman et al. (ref. 5).
C. Platteeuw and W. M. de Vos (1992, ref. 3) described the location, characterization and expression in Escherichia coli of lytic enzyme-encoding gene, lytA, of Lactococcus lactis bacteriophage .phi.US3. It was described that the .phi.vML3 lysin, which is active on a wide range of lactococcal strains, lacked homology with known lytic enzymes. The bacteriophage .phi.US3 was identified during studying bacteriophages specific for the cheese-making strain Lactococcus lactis SK11 (NIZO). The results showed that the deduced amino acid sequence of LytA shares similarities with that of an autolysin of Streptococcus pneumonia, suggesting that the bacteriophage .phi.US3 encodes an amidase rather than a lysozyme-type muramidase. The above illustrates the difficulties facing a person skilled in the art wishing to isolate DNA-sequences from different organisms. The lack of information regarding sequences and the lack of homology between known sequences makes use of probes and primers derived from known sequences quite unlikely to lead to successful isolation of a correct DNA sequence from different organisms.
In EP-A2-0 510 907 (AFRC, M. J. Gasson, published Oct. 28, 1992, ref. 7) the use of bacteriophages of food-contaminating or pathogenic bacteria or the lysins thereof to kill such bacteria was described. Examples included lysins from bacteriophages of Listeria monocytogenes (phage .phi.LM4) and Clostridium tyrobutyricum (phage .phi.P). Also tests for bacterial contamination can be made specific for specific bacteria by using the appropriate bacteriophage or lysin thereof and determining whether cells are lysed thereby. That European patent application thus describes the use of lysins obtained from phages of food-contaminating or even pathogenic bacteria, which is not desirable for food-grade applications. Moreover, the purpose of use of such lysins in that patent application is further away from the subject of this invention, which will be discussed below as it does not lie in improving flavour of food products by autolysis of lactic acid bacteria.